Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Adjuvants are included or added to vaccines and other immunogenic formulations, to increase and in some cases direct the immune response (see reviews by Gupta, 1998, Cox and Coulter, 1992, Azuma, 1992 and Aguado et al, 1999). It will be appreciated that some adjuvants, for example the oil in water and water in oil emulsions, are widely considered as strong adjuvants as they stimulate high levels of antibody, but the reactogenicity of such adjuvants precludes their routine use in many animal species or in man. The unacceptable reactogenicity has been demonstrated in many animals with the traditional Freunds' oil in water emulsion, to the extent that the use of this adjuvant in animals has been prohibited or discouraged in some countries, even for experimental purposes. The use of a combined oil in water/DEAE-dextran adjuvant in cattle (Hodgkinson et al, 1990) resulted in significant and undesirable reactions in 30% of the vaccinated animals.
The most widely used adjuvants in man and animals are based on insoluble aluminium salts, particularly the hydroxide and phosphate forms, commonly and collectively termed “alum”. The recent review by Aguado et al (1999) highlights the ongoing need for stronger adjuvants in both man and animals for the development of more effective vaccines.
For example, with a wide range of soluble antigens, the immune response to such an antigen delivered alone is very poor, and in many cases is undetectable even after two vaccinations. Such antigens require the use of an adjuvant to stimulate a consistent immune response. It will be appreciated that known adjuvants offer a range of abilities to stimulate an immune response, most usually defined in terms of antibody response to the immunising antigen.
For haptens coupled to a carrier protein, it is widely recognised that a powerful adjuvant is required. For example, the hypothalmic peptide hormone LHRH is a 10 amino acid peptide that by itself is non-immunogenic. Coupling of LHRH to carrier proteins, for example diphtheria toxoid or ovalbumin provides the necessary T-cell help for an immune response to the LHRH hapten. For other large molecular weight and complex antigens, the T cell help is usually provided by T-cell epitopes within the molecule itself.
The use of DEAE-dextran as an adjuvant has been well described in the scientific literature. Wittman et al (1975), used DEAE-dextran to adjuvant a Foot and Mouth Virus (FMDV) vaccine in pigs, and showed that it induced very high levels of immunity. Houston et al (1976) used this adjuvant at a dose rate of 1-5 mg/kg body weight, to vaccinate monkeys with an equine virus and showed that it induced high titres of antibody. Beh and Lascelles (1985) have also used this adjuvant to immunise sheep with ovalbumin, and showed that it induced higher antibody titres than other soluble adjuvants. None of these studies considered the site reactivity of DEAE-dextran, but they did describe the significant adjuvant effects of DEAE-dextran in these animal species. DEAE-dextran has been used in deer with keyhole limpet haemocyanin (KLH) antigen (Hibma and Griffin, 1992). In this study, DEAE-dextran induced greater IgG responses to KLH than did Freunds adjuvant or alum.
The adjuvant properties of saponin have been long known, as has its ability to increase antibody titres to immunogens. As used herein, the term “saponin” refers to a group of surface-active glycosides of plant origin composed of a hydrophilic region (usually several sugar chains) in association with a hydrophobic region of either steroid or triterpenoid structure. Although saponin is available from a number of diverse sources, saponins with useful adjuvant activity have been derived from the South American tree Quillaja saponaria (Molina). Saponin from this source was used to isolate a “homogeneous” fraction denoted “Quil A” (Dalsgaard, 1974).
Dose-site reactivity is a major concern for both the veterinary and human use of Quil A in vaccine preparations. One way to avoid this toxicity of Quil A is the use of immunostimulating complexes (known as Iscoms™, an abbreviation for Immuno Stimulating COMplexes). This is primarily because Quil A is less reactive when incorporated into immunostimulating complexes, because its association with cholesterol in the complex reduces its ability to bind to cholesterol in cell membranes and hence its cell lytic effects. In addition, a lesser amount of Quil A is required to generate a similar level of adjuvant effect.
The immunomodulatory properties of the Quil A saponins and the additional benefits to be derived from these saponins when they are incorporated into an immunostimulating complex have been described in various publications, e.g. Cox and Coulter, 1992; Dalsgaard, 1974; Morein et al., Australian Patent Specifications Nos. 558258, 589915, 590904 and 632067.
Vaccination against the hypothalamic hormone luteinising hormone releasing hormone (referred to herein as “LHRH”, also known as GnRH) has been demonstrated as an immunological method of controlling reproduction since the early 1970's (Fraser 1975, Jeffcoate et al 1982). Eliciting an immune response to LHRH prevents the release from the anterior pituitary of the hormones LH and FSH, which are required for the development and maintenance of the gonads—the testes in the male and ovaries in the female. Thus reduction of LH and FSH levels leads to loss of reproductive function.
De-sexing (or neutering) operations are the most widely practised surgical procedures in veterinary medicine and livestock animal management. A significant proportion of both sexes of domestic livestock and companion animals are routinely surgically de-sexed to prevent a variety of undesirable characteristics which accompany sexual maturity. The traits include fighting, wandering, sexual behaviour, loss of condition, tumours of reproductive organs and pregnancy.
The control of mating behaviour by vaccination with LHRH-conjugate vaccines in companion animals such as dogs, cats and horses, and in livestock specifically in male pigs and male and female cattle, has been identified as a goal as significant as the control of fertility.
Similarly, the control and treatment of disorders of the gonads and other reproductive organs, of both humans and animals, such as testicular cancer, breast cancer, prostate cancer, ovarian cancer, prostate enlargement or endometriosis is of significance.
International Patent Application No. PCT/AU98/00532, the contents of which are incorporated herein by reference, discloses that the efficacy of vaccination against LHRH is significantly improved when LHRH is administered as a conjugate with diphtheria toxoid and an ionic polysaccharide such as DEAE-dextran. However, although the efficacy is improved, this formulation nevertheless induces reactogenicity such as visible swelling, that may belong-lasting, at the site of administration. In some instances, such as where the formulation is used to vaccinate domestic pets (for example, dogs) or livestock destined for consumption the occurrence of such reactogenicity following vaccination against LHRH utilising this formulation is unacceptable. Accordingly, there is a need to develop a LHRH vaccine which exhibits both efficacy and low reactogenicity.
In one aspect of the work leading up to the present invention, it has been determined that reactogenicity of LHRH conjugated to diphtheria toxoid and an ionic polysaccharide can be reduced when a proportion of the ionic polysaccharide component is replaced with a saponin component, particularly an immunostimulating complex.